Possession of a nervous system endows an organism with a substantial range of behavioral abilities owing to a diverse class of excitatory cells in the brain termed neurons and, more importantly, the connections between them. Primary sensory information flows through neural circuits, being shaped and processed as it does, resulting in perception, thought, decision, and action. The processing power of any individual neuron, while considerable, cannot account for such complex behavior. Rather, it is an emergent property of an interconnected system of neurons, the primary unit of connection being the chemical synapse. These synapses permit fast, moment-to-moment transmission of information between cells without compromising the electrical integrity of an individual cell, allowing integration of information can take place at both a cellular and circuit level. Healthy connections allow for split-second decisions, breathtaking insights, and incredible feats of coordination, whereas disrupted transmission can lead to devastating diseases such as schizophrenia and autism. These synaptic sites of connection between neurons are anatomical as well as functional - requiring an initial physical interaction between cells in the process of establishing a meaningful connection. This thesis begins with an inquiry into these anatomical aspects of a synapse. Specifically, I present evidence for the requirement of synaptic adhesion molecules in the formation and plasticity of synapses. I take one family of postsynaptic adhesion molecule, neuroligin, as a model for the class to test its specific function with regard to synaptogenesis and plasticity. I identify and describe three major functions of the protein, each dependent on a specific structural component: one, differentiation of the presynaptic terminal, dependent on dimerization, achieved via the trans-synaptic clustering of the presynaptic adhesion molecule neurexin; two, recruitment of postsynaptic components during synaptic assembly, dependent on a previously uncharacterized region of the postsynaptic intracellular domain, achieved via the scaffolding of the postsynaptic site; and three, specification of synaptic subtype, dependent on alternative splicing of the extracellular domain, achieved via a trans-synaptic protein binding code. Finally, I examine the distribution of synapses along a dendritic arbor and the determination of relative synaptic weight as it relates to the electrical integrative properties of a cell.

Merkel cell-neurite complexes are highly sensitive touch receptors comprising sensory afferents and epidermal Merkel cells. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To ask whether Merkel cells are genetically programmed to be excitable cells, we purified Merkel cells from touch domes and used DNA microarrays to compare gene expression in Merkel cells and other epidermal cells. We identified 362 Merkel-cell-enriched transcripts including neuronal transcription factors, presynaptic molecules and ion-channel subunits. Antibody staining of skin sections showed that Merkel cells are immunoreactive for presynaptic proteins, including piccolo, Rab3C, VGLUT2 and cholecystokinin. These data indicate that Merkel cells are poised to release glutamate and neuropeptides. Finally, using Ca2+ imaging, we discovered that Merkel cells have L-type and P/Q-type voltage-gated Ca2+ channels, which have been shown to trigger vesicle release at synapses.

We also asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and direct indentation with probes while responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in actin-filled processes. Third, voltage activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ induced Ca2+ release amplifies signals from swelling activated cation channels. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of Merkel-cell hypotonic responses. Collectively, these results directly demonstrate that Merkel cells are mechanosensitive, identify cellular signaling mechanisms that mediate mechanically evoked responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex.

Excitation/inhibition balance is critical for optimal brain function, yet the mechanisms underlying the tuning of inhibition from different populations of inhibitory neurons are unclear. Here, we found evidence for two distinct pathways through which excitatory neurons cell-autonomously modulate inhibitory synapses. Synapses from parvalbumin-expressing interneurons onto hippocampal pyramidal neurons are regulated by neuronal firing, signaling through L-type calcium channels. Synapses from somatostatin-expressing interneurons are regulated by NMDA receptors, signaling through R-type calcium channels. Thus, excitatory neurons can cell-autonomously regulate their inhibition onto different subcellular compartments through their input (glutamatergic signaling) and their output (firing). We found that in both the regulation of parvalbumin synapses by action potentials, and the regulation of somatostatin synapses by NMDA receptors represent alterations in the number of functional synapses.

Separately, little is known regarding whether synapses formed by somatostatin and parvalbumin interneurons onto pyramidal neurons are composed of different sets of post-synaptic components. Here, we found that while somatostatin and parvalbumin synapses onto excitatory neurons are both dependent on a common set of post-synaptic proteins, including gephyrin, collybistin, and neuroligin-2, somatostatin synapses are selectively impacted by neuroligin-3. Decreasing neuroligin-3 expression selectively decreases inhibition from somatostatin interneurons, and overexpression of neuroligin-3 selectively enhances somatostatin inhibition. Together, these results provide evidence that excitatory neurons can selectively regulate two distinct sets of inhibitory synapses.

Memories are stored in the brain via specific patterns of connectivity between individual neurons. Learning occurs through changes in this pattern of connectivity in response to activity, such that a synapse becomes more or less effective at influencing a postsynaptic neuron. This process, called synaptic plasticity, has been demonstrated at excitatory glutamatergic synapses of the hippocampus, where precise patterns of activity can either increase (long-term potentiation, LTP) or decrease (long-term depression, LTD) synaptic strength. Both LTP and LTD are carried out through changes in the number of postsynaptic AMPA-type glutamate receptors (AMPARs). Therefore, to understand synaptic plasticity, we must also understand the trafficking of AMPARs. In the case of LTP expression, the AMPAR subunit GluA1 is specifically required, and modifications of its cytoplasmic tail (C-tail) are thought to be particularly important for the activity-dependent recruitment of AMPARs. To identify the minimum region of the GluA1 C-tail required for LTP, I used a single-cell molecular replacement strategy where all endogenous AMPARs are replaced with transfected subunits. Surprisingly, I found no requirement for the GluA1 C-tail or for GluA1 generally for expression of LTP. Instead, molecular replacement with either GluA2 or the kainate receptor subunit GluK1 resulted in normal LTP. The only conditions under which LTP was impaired were those with a dramatically decreased pool of receptors on the neuronal surface. Similar to LTP, I also found no specific AMPAR subunit requirement for LTD, which was expressed normally in neurons only expressing GluK1. These results suggest that synaptic plasticity is not necessarily a direct modification of the glutamate receptors subunits themselves, but a broader change in the ability of the synapse to anchor postsynaptic receptors.

Ion channels are often modulated by auxiliary subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary subunits for AMPA-type glutamate receptors. These receptors are responsible for much of the fast excitatory synaptic transmission in the brain, and their mobility is thought to contribute to memory storage mechanisms. Although TARPs are known to modify AMPA receptor trafficking and gating in vitro, their contribution to in vivo AMPA receptor function is less clear.

By generating mice lacking multiple TARP family members, we found that TARPs are functionally redundant. Single TARP knockout mice are viable, while those lacking multiple isoforms are often lethal. Consistent with molecular redundancy, AMPA receptor transmission in cerebellar Golgi cells is unaffected in single TARP knockout mice, but nearly eliminated in double knockouts. Unexpectedly, the remaining AMPA receptors have a different subunit composition, suggesting that TARPs may preferentially traffic GluR2 containing receptors.

Our studies also highlight a role for TARPs in inhibitory neurons because AMPA receptor function in cerebellar Purkinje cells and Golgi cells is reduced in TARP knockout mice. Additionally, the loss of TARPs reduces the decay time of interneuron EPSCs. This indicates that AMPA receptor biophysical properties make a significant contribution to the time course of synaptic events, even at fast-decaying interneuron synapses.

Our studies investigating TARP function in vivo also led to the discovery that TARPs profoundly change AMPA receptor pharmacology. Whereas CNQX is a competitive antagonist on AMPA receptors alone, it is a partial agonist on receptors containing any member of the TARP family and elicits depolarizing currents in neurons throughout the brain. By obtaining the crystal structure of CNQX bound to the AMPA receptor ligand-binding domain, we determined that CNQX induces a small amount of domain closure. Based on our findings, we propose an expanded model of AMPA receptor gating such that TARPs increase the likelihood that domain closure leads to channel opening, i.e. they increase the coupling efficiency. Together our studies demonstrate that TARPs are an integral component of AMPA receptors in the central nervous system.